BR112020000274A2 - new fab fragment of human anti-ceacam5 antibody - Google Patents

Abstract

A Fab fragment of the human anti-CEACAM5 antibody, which can be useful in the diagnosis of cancer, particularly in the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from the metastasis thereof, as well as a diagnostic approach using a conjugate comprising the Fab fragment are provided. The present invention features a Fab fragment of the human anti-CEACAM5 antibody, which comprises a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2, and a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4, and a conjugate comprising the Fab fragment.

Description

“NEW FAB FRAGMENT OF THE HUMAN ANTI-CEACAM5 ANTIBODY”

TECHNICAL FIELD

[001] The present invention relates to a new Fab fragment of the human anti-CEACAM5 antibody. The present invention also relates to a diagnostic composition comprising the Fab fragment of the human anti-CEACAM5 antibody, and a method for diagnosing cancer by using the Fab fragment.

STATE OF ART

[002] CEA (carcinoembryonic antigen) or CEACAM (cell adhesion molecule related to carcinoembryonic antigen) is a tumor marker discovered in 1965 (J. Exp. Med .; 1965; 121: 439-462; and PNAS; 1969; 64 : 161-167) and 23 molecules related to CEA have been revealed so far (BioMed Central Biology; 2010; 8: 12-33). Among them, CEACAM5 is rarely expressed in normal tissues, but it is expressed in the fetal gastrointestinal tract or in colorectal cancer (BBA; 1990; 1032: 177-189; and Mol.

Pathol .; 1999; 52: 174-178). CEACAM5 is known to be expressed also in breast cancer, lung cancer and thyroid cancer (Diagn. Cytopathol .; 1993; 9: 377-382; Cancer Res .; 1990; 50: 6987-6994; Histopatology; 2000; 37: 530-535).

[003] The concentration of CEACAM5 in the blood is higher in patients with colorectal cancer than in healthy people (J. Exp Med .; 1965; 121: 439-462), and CEACAM5 is used as a tumor marker. According to histological studies of patients with colorectal cancer, CEACAM5 is highly expressed in 90% or more of the tissues (British J. Cancer; 2013; 108: 662-667).

[004] Since early colorectal cancer metastasis is confined to the liver, early detection and treatment of liver metastases can reduce recurrence rates (Cell Mol Gastroenterol Hepatol .; 2017; 3: 163-173). The diagnosis of liver metastasis uses CT (computed tomography), MRI (magnetic resonance imaging), or FDG-PET (positron emission tomography with fluorodeoxyglucose). The detection sensitivity of CT, RMI, and FDG-PET is 74.4, 80.3, and 81.4%, respectively, and the detection sensitivity of tumors of 1 cm or smaller is reduced to 47.3% for CT and 60.2% for RMI (Radiology, 2010; 257: 674-684). MRI using liver-specific contrast agents is also applied, although its sensitivity for detecting tumors of 1 cm or less is 29 to 38% (Radiology; 2005; 237: 89-98).

[005] Anti-cancer agents or antibodies marked with a metal radioisotope are used for the diagnosis or treatment of cancers. Antibody targeting has high specificity for tumor cells and causes fewer adverse events. Some monoclonal antibodies marked with a metallic radioisotope have been clinically applied to diagnosis and treatment so far (Cancer Control; 2012; 19: 196-203).

[006] On the other hand, antibodies generally have a long half-life in the blood and require a period as long as 4 days to 5 days to reach a tumor: blood ratio that gives a sufficient signal: background ratio for the visualization of a cancer after administration to the body (Clin.

Pharmacol. The R; 2010; 87: 586-592). In addition, the antibody Fc regions cause an pharmacological effect of antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (Glycoconj J .; 2013; 30: 227-236; and Curr Opin Biotechnol .; 2002; 13: 609-614).

In addition, antibodies are metabolized in the liver and therefore accumulate in the liver, regardless of the target. However, the detection of liver metastasis lesions with the use of antibodies is complex, since early colorectal cancer metastases are confined to the liver (Clin. Pharmacol. Ther; 2010; 87; 586-592).

[007] Fragments of recombinant antibodies of low molecular weight, such as Fab, scFv, diabody, reach the lesions easily due to their high penetration into the tissue and are used as therapeutic antibodies, since the production that uses an expression system in E. coli or yeast can be inexpensive. On the other hand, its use as a diagnostic drug has also been reported because of its short half-lives in the blood and the characteristic of renal excretion (Nat. Biotechnol .; 2005; 23: 1126-1136).

[008] An M5A (PTL 1), a humanized antibody to the murine monoclonal antibody T84.66, is known as a human anti-CEACAM5 antibody applied as a diagnostic drug. 64Cu-labeled M5A has been shown to require an interval of 22 hours or more after administration to obtain favorable PET images in a test with mice transplanted subcutaneously with cancer cells (NPL 1), and is absorbed into normal tissue from the liver and a liver injury site, at the same level, 3 hours after administration and with a significant difference after a 24-hour interval in a test using models in mice with liver metastases (NPL 2).

[009] As for a fragment of a human anti-CEACAM5 antibody, it was shown that CEA-Scan, Fab 'of the 99mTc-labeled mouse monoclonal antibody NP-4, can be used in the diagnosis of colorectal cancer (NPL 3) . However, the uptake of CEA-Scan at an injury site is not greater than its uptake in normal liver, and its detection sensitivity for liver metastases is less than that of FDG-PET (NPL 4). CEA-Scan was approved as a diagnostic drug for colorectal cancer by the FDA in 1999, but is no longer sold (NPL 5).

CITATION LIST

PTL 1 PATENTARY LITERATURE: International Publication No. WO 2005/086875

NENT PATENTARY LITERATURE NPL 1: Bioconjug. Chem .; 2008; 19: 89-96 NPL 2: PLOS ONE; 2014; 9 (9): e106921 NPL 3: Ann. Surg .; 1997; 226: 621-631 NPL 4: J. Nucl. Med .; 2000; 41: 1657-1663 NPL 5: Kenneth T. Cheng, "99mTc-Arcitumomab", [online], Update: March 17, 2008., Molecular Imaging and Contrast Agent Database, [search on May 17, 2017], website <URL: https://www.ncbi.nlm.nih.gov/books/NBK23676/>

SUMMARY OF THE INVENTION TECHNICAL PROBLEM

[010] Monovalent Fab fragments have a molecular weight of approximately 50 KDa, which is less than (approximately 150 kDa) of antibodies, undergo renal excretion, and also have a short half-life in the blood. Thus, they achieve a tumor: blood ratio that gives a signal: background ratio sufficient to be able to detect liver metastases and visualize cancer within 2 to 32 hours after administration. They do not have an Fc region and therefore do not cause ADCC or CDC. In view of these characteristics, Fab fragments are expected to be more effective as diagnostic drugs compared to antibodies.

[011] However, the binding activity of the Fab fragments is often attenuated because it is a small molecule. Antibodies should be labeled with a detectable substance, such as a PET tracer or fluorescent dye for their use as in vivo diagnostic drugs. Another problem with Fab fragments is the attenuation of their binding activity due to labeling with such a substance.

[012] An object of the present invention is to provide a Fab fragment of the anti-human CEACAM5 antibody which is useful in the detection of human CEACAM5 and can accumulate in a cancer lesion within a short period of time (for example, 4 hours) after administration. Another object of the present invention is to provide a diagnostic composition comprising the Fab fragment that can enable diagnosis on the day of administration, and a diagnostic method using it.

SOLUTION TO THE PROBLEM

[013] The present inventors have carried out considerable diligent studies on the preparation of a Fab fragment of the human anti-CEACAM5 antibody useful in the detection of human CEACAM5, and, consequently, have prepared a Fab fragment of the human anti-CEACAM5 antibody comprising a variable region of the heavy chain consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 and a variable region of the light chain consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID: NO: 4 (Example 1) and found that: the Fab fragment of the anti-human CEACAM5 antibody does not attenuate the binding activity against human CEACAM5 by binding a labeling portion (Example 3); and a conjugate comprising the Fab fragment of the anti-human CEACAM5 antibody accumulates in CEACAM5 positive human cancer cells in subcutaneous transplant models and liver transplant model 4 hours after administration and allows the detection of CEACAM5 positive human cancer cells ( Examples 5 and 6). Specifically, the present invention provides a Fab fragment of the human anti-CEACAM5 antibody that accumulates in a cancer, and a conjugate comprising the Fab fragment of the human anti-CEACAM5 antibody.

[014] The present invention includes aspects presented below as substances and methods that are medically or industrially useful.

[015] Specifically, in one aspect, the present invention may be as follows: (1) A Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a variable region of the heavy chain comprising CDR1 consisting of a sequence of amino acids from amino acid positions 31 to 35 of SEQ ID NO: 2, CDR2 consisting of a sequence of amino acids from amino acid positions 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of a sequence of amino acids from amino acid positions 99 to 110 of SEQ ID NO: 2, and a light chain comprising a variable region of the light chain comprising CDR1 consisting of an amino acid sequence from amino acid positions 24 to 38 of SEQ ID NO: 4, CDR2 consisting of an amino acid sequence from amino acid positions 54 to 60 of SEQ ID NO: 4 and CDR3 consisting of an amino acid sequence from amino acid positions 93 to 101 of SEQ ID NO: 4.

(2) The Fab fragment of the human anti-CEACAM5 antibody, according to (1), selected from the group consisting of the following items (a) and (b): (a) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a variable region of the heavy chain consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 and a light chain comprising a variable region of the light chain consisting of a sequence of amino acids from amino acid positions 1 to 112 of SEQ ID NO: 4; and (b) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a heavy chain variable region derived from a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 by modification of glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid and a light chain comprising a variable region of the light chain consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4. (3) The Fab fragment of the human anti-CEACAM5 antibody, according to (2), is selected from the group consisting of the following items (a) and (b): (a) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4; and (b) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment derived from a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 by modifying glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

(4) The Fab fragment of the human anti-CEACAM5 antibody according to (3), comprising a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the represented amino acid sequence by SEQ ID NO: 4.

(5) A conjugate comprising a labeling portion and a Fab fragment of the human anti-CEACAM5 antibody, according to any one of items (1) to (4). (6) The conjugate according to (5), wherein the labeling portion is (i) a linker and a linker, (ii) a linker, (iii) a fluorescent dye and a linker, or ( iv) a fluorescent dye.

(7) The conjugate according to (6), wherein the labeling portion is (i) a linker and a linker, or (ii) a linker.

(8) The conjugate according to (7), where the linker is a linker represented by the following formula (A): [Chemical formula 1] (A) where the wavy line represents the binding to the Fab fragment of the anti- Human CEACAM5 or the binding agent.

(9) The conjugate according to (7), wherein the labeling portion is (i) a linker and a linker, where the linker and the linker are a group represented by the following formula (A ') : [Chemical formula 2]

(A ') where the wavy line represents the binding to the Fab fragment of the human anti-CEACAM5 antibody.

(10) The conjugate according to (9), wherein the Fab fragment of the human anti-CEACAM5 antibody is attached to the carbon atom of a terminal C (= S) group of the labeling portion via an amino group on the Fab fragment .

(11) The conjugate according to (7) represented by the following formula (I): (L-X) p-Ab (I) where Ab is the Fab fragment of the human anti-CEACAM5 antibody; L is the ligand; X is the bonding agent or a bond; and p is a natural number from 1 to 25. (12) The conjugate according to (11), where L is a linker represented by the following formula (A): [Chemical formula 3]

(A) where the wavy line represents the bond to X (or Ab when X is a bond).

(13) The conjugate according to (9) or (12) represented by the following formula (II): [Chemical formula 4] (II) p where Ab is the Fab fragment of the human anti-CEACAM5 antibody; and p is a natural number from 1 to 25, where Ab is attached to the terminal group C (= S) carbon atom of the labeling portion through an amino group on Ab.

(14) The conjugate according to any of items (11) to (13), where p is a natural number from 1 to 16.

(15) The conjugate according to any of (11) to (13), where p is a natural number from 4 to 16.

(16) The conjugate according to any one of (6) to (15), further comprising a metal.

(17) The conjugate according to (16), in which the metal is a metallic radioisotope.

(18) The conjugate according to (16), in which the metal is 89Zr. (19) The conjugate according to (17) or (18) for use as a PET tracer.

(20) A polynucleotide selected from the group consisting of the following items (a) and (b): (a) a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody (a) of according to (2); and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2).

(21) A polynucleotide selected from the group consisting of the following items (a) and (b): (a) a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody according to (4); and (b) a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4).

(22) An expression vector comprising the following items (a) and / or (b):

(a) a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) according to (2); and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2).

(23) An expression vector comprising the following items (a) and / or (b): (a) a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody according to (4); and (b) a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4).

(24) A host cell selected from the group consisting of the following items (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the fragment's heavy chain fragment Fab of human anti-CEACAM5 antibody (a) according to (2); (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2); (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of the anti-antibody Fab fragment

Human CEACAM5 (a) according to (2) and a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2); and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) according to (2) and an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2).

(25) A host cell selected from the group consisting of the following items (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody according to (4); (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4); (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment according to (4) and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4); and

(d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment according to (4) and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4).

(26) Method for producing a Fab fragment of the human anti-CEACAM5 antibody, comprising the step of culturing a host cell selected from the group consisting of the following items (a) to (c) to express the Fab fragment of the anti antibody -CEACAM5 human: (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the anti-human CEACAM5 antibody (a) according to (2) and a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2); (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) according to (2) and an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2); and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) according to (2) and a transformed host cell with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) according to (2).

(27) A method for producing a Fab fragment of the human anti-CEACAM5 antibody, comprising the step of culturing a host cell selected from the group consisting of the following items (a) to (c) to express the Fab fragment of the human anti-CEACAM5 antibody: (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody according to (4) and a polynucleotide containing a nucleotide sequence that encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4); (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment according to (4) and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4); and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment according to (4) and a host cell transformed with a vector of expression comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody according to (4).

(28) Method for the production of a conjugate comprising a labeling portion and a Fab fragment of the human anti-CEACAM5 antibody, comprising the steps of: preparing the Fab fragment of the human anti-CEACAM5 antibody by a method according to (26) or (27); and covalently attaching the Fab fragment to the tag portion.

(29) Method for the production of a conjugate comprising a ligand and a Fab fragment of the human anti-CEACAM5 antibody, comprising the steps of: preparing the Fab fragment of the human anti-CEACAM5 antibody by a method according to (26) or ( 27); and covalently attaching the Fab fragment to the linker via a linker or directly.

(30) Method for the production of a conjugate comprising a labeling portion labeled with a metal radioisotope and a Fab fragment of the human anti-CEACAM5 antibody, comprising the steps of: producing a conjugate comprising a ligand and a Fab fragment of the anti-human antibody Human CEACAM5 by a method according to (29); and connecting the metal radioisotope to the conjugate ligand through a coordinated connection.

(31) A diagnostic composition comprising a conjugate according to any one of (16) to (19) and a pharmaceutically acceptable carrier.

(32) The diagnostic composition according to (31), which is a drug for clinical staging.

(33) The diagnostic composition according to (31) or (32) for use in the diagnosis of colorectal cancer, breast cancer, lung cancer, thyroid gland cancer or a cancer resulting from their metastasis.

(34) The diagnostic composition according to (33), for use in the diagnosis of colorectal cancer or a cancer resulting from colorectal cancer metastasis.

(35) The diagnostic composition according to (34), in which the cancer resulting from colorectal cancer metastasis is metastatic liver cancer.

(36) Use of a conjugate according to (16) to (19) for the production of a composition for the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from their metastasis .

(37) The conjugate according to any of (16) to (19) for use in the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from their metastasis.

(38) A method for diagnosing colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or cancer resulting from metastasis thereof, comprising administering a conjugate according to (16) to (19) to an individual .

(39) The Fab fragment of the human anti-CEACAM5 antibody, according to any one of (1) to (4), for use as a conjugate comprising a labeling portion.

(40) Use of a Fab fragment of the human anti-CEACAM5 antibody, according to any one of (1) to (4), for the production of a conjugate comprising a labeling portion.

(41) The Fab fragment of the human anti-CEACAM5 antibody according to (39), wherein the conjugate comprises a labeling portion is a conjugate according to any one of (5) to (18).

(42) The use of a Fab fragment of the human anti-CEACAM5 antibody according to (40), wherein the conjugate comprises a labeling portion is a conjugate according to any one of (5) to (18).

ADVANTAGE EFFECTS OF THE INVENTION

[016] The Fab fragment of the human anti-CEACAM5 antibody of the present invention is useful for the detection of human CEACAM5 and may be useful in the diagnosis of a cancer, such as colorectal cancer, breast cancer, lung cancer, gland cancer thyroid or a cancer resulting from their metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[017] [FIG. 1A] Fig. 1A is a representative image obtained by PET 4 hours after administration of PB009-03 to a mouse in which cells of the human colorectal cancer cell line - LS174-T (human cells positive for CEACAM5) were transplanted subcutaneously on the right shoulder, while cells of the human colorectal cancer cell line - HCT-15 (human cells negative for CEACAM5) were transplanted subcutaneously on the left shoulder. Since the mouse was photographed face down, the right circle shows the right shoulder of the mouse transplanted with the LS174T cells and the left circle represents the left shoulder of the mouse transplanted with the HCT-15 cells. The right bar represents a maximum standardized uptake value (SUV-Max) by the tumor.

[FIG. 1B] Fig. 1B is a graph illustrating the results of the analysis of images obtained by PET 4 hours, 24 hours and 48 hours after the administration of PB009-03 to a mouse, in which the cells of the human colorectal cancer cell line - LS174T (human cells positive for CEACAM5) were transplanted subcutaneously in the right shoulder, while cells of the human colorectal cancer cell line - HCT-15 (human cells negative for CEACAM5) were transplanted subcutaneously in the left shoulder. The ordinate represents an SUV-Max value of PB009-03 that has accumulated at the tumor site. The error bars in the graph represent mean ± standard deviation (mean ± SEM).

[FIG. 2A] Fig. 2A is a representative image obtained by PET 4 hours after administration of PB009-03 to a mouse, in which the LS174T cells expressing luciferase (human cells positive for CEACAM5) were transplanted into the liver. The arrow shows a transplanted site with the cells.

The right bar represents an SUV-Max value. [FIG. 2B] Fig. 2B is a graph illustrating the results of the analysis of the images obtained by PET 4 hours and 24 hours after administration of PB009-03 to a mouse, in which the LS174T cells expressing luciferase (human cells positive for CEACAM5 ) were transplanted in the liver. The ordinate represents a ratio between the SUV-Max values of PB009-03 that have accumulated in the liver and in a tumor site (tumor / liver ratio). The error bars in the graph represent mean ± standard deviation (mean ± SEM).

DESCRIPTION OF METHODS

[018] The present invention will be described in detail hereinafter. However, the present invention is not limited by them. Scientific terms and technical terms used in connection with the present invention have meanings that are generally understood by those skilled in the art, unless otherwise specified.

[019] The present inventors have carried out considerable diligent studies on the preparation of a human anti-CEACAM5 antibody or an antigen-binding fragment thereof and, consequently, have successfully prepared a Fab fragment of the human anti-CEACAM5 antibody useful for the detection of Human CEACAM5.

[020] The basic structure of an antibody molecule is common across classes and consists of heavy chains having a molecular weight of 50000 to 70000 and light chains having a molecular weight of 20000 to 30000. The heavy chain generally consists of a chain polypeptide comprising about 440 amino acids, has a structural characteristic of each class, and is called γ, µ, α, δ, and ε chains corresponding to IgG, IgM, IgA, IgD and IgE. IgG also has IgG1, IgG2, IgG3 and IgG4, which are called γ1, γ2, γ3 and γ4, respectively. The light chain generally consists of a polypeptide chain comprising about 220 amino acids and is known as two types, types L and K, which are called λ and κ chains, respectively. As for the peptide configuration of the basic structure of the antibody molecule, two homologous heavy chains and two homologous light chains are linked by disulfide bonds (SS bonds) and non-covalent bonds to form a molecular weight of 150000 to 190000. The two light chains can match with any of the heavy chains. An individual antibody molecule is constantly made up of two identical light chains and two identical heavy chains.

[021] Four (or five for µ and ε chains) and two intrachain SS bonds are present in the heavy chain and the light chain, respectively, and each constitutes a loop or loop for 100 to 110 amino acid residues. This conformation is similar between loops and is called a structural domain or unit. For the heavy chain and light chain, a domain positioned on the N-terminal side does not have a constant sequence of amino acids, even between preparations of the same classes (subclasses) of animals of the same species and, therefore, is called a variable region. The respective domains are called the heavy chain variable region (VH domain) and the light chain variable region (VL domain). An amino acid sequence on the C- side

terminal is almost constant in relation to the class or subclass and is called a constant region. The respective domains are represented by CH1, CH2, CH3 and CL.

[022] The specificity of binding the antibody to an antigen depends on the amino acid sequence of a fraction consisting of the variable region of the heavy chain and the variable region of the light chain. On the other hand, biological activity, such as binding to complements or several cells, reflects the difference in structure between the regions contained in the Igs of the respective classes. It is known that the variability of the variable regions of the heavy and light chain is substantially limited by three small hypervariable regions present in both chains. These regions are called complementarity determining regions (CDRs; CDR1, CDR2 and CDR3 in order from the N-terminal side). The remaining portions of the region are called structural regions or frameworks (FRs) and are relatively constant.

[023] A region between the CH1 domain and the CH2 domain of an antibody heavy chain constant region is called a hinge region.

This region is rich in proline residues and contains a plurality of S-S interchain links that connect two heavy chains. For example, the hinge regions of IgG1, IgG2, IgG3 and IgG4 contain 2, 4, 11, and 2 cysteine residues, respectively, which constitute the S-S bonds between the heavy chains. The hinge region is a region highly sensitive to a proteolytic enzyme, such as papain or pepsin. In the case of digestion of an antibody with papain, the heavy chains are cleaved at a position on the N-terminal side of the heavy S-S linkings of the hinge region and thus decomposed into two Fab fragments and an Fc fragment. The Fab fragment is made up of a light and heavy chain fragment comprising a variable region of the heavy chain, a CH1 domain and a portion of the hinge region. The Fab fragment comprises variable regions and has antigen-binding activity.

Fab fragment of the human anti-CEACAM5 antibody of the present invention

[024] The Fab fragment of the human anti-CEACAM5 antibody of the present invention includes a Fab fragment having the following characteristic: a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a variable region of the heavy chain consisting of a amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 and a light chain comprising a variable region of the light chain consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4.

[025] Any Igγ1, Igγ2, Igγ3 or Igγ4 constant region, etc., can be selected as the heavy chain constant region of the Fab fragment of the human anti-CEACAM5 antibody of the present invention. In one embodiment, the heavy chain constant region of the human anti-CEACAM5 antibody Fab fragment of the present invention is a human Igγ1 constant region.

[026] Any Igλ or Igκ constant region can be selectable as the light chain constant region of the Fab fragment of the human anti-CEACAM5 antibody of the present invention. In one embodiment, the light chain constant region of the Fab fragment of the human anti-CEACAM5 antibody of the present invention is a human Igκ constant region.

[027] In one embodiment, the Fab fragment of the human anti-CEACAM5 antibody of the present invention is the following Fab fragment: a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[028] In the case of expression of an antibody including a Fab fragment in cells, the antibody is known to undergo post-translational modification.

Examples of post-translational modification include cleavage of heavy chain C-terminal lysine by carboxypeptidase, modification of glutamine or N-terminal heavy chain and light chain glutamic acid in pyroglutamic acid by pyroglutamylation, glycosylation, oxidation, deamidation and glycation . Such post-translational modification is known to occur in several antibodies (J. Pharm Sci, 2008; 97: 2426-2447).

[029] The Fab fragment of the human anti-CEACAM5 antibody of the present invention also includes a Fab fragment resulting from post-translational modification. Examples of the Fab fragment of the human anti-CEACAM5 antibody of the present invention that can result from post-translational modification include a Fab fragment of the human anti-CEACAM5 antibody containing an N-terminal pyroglutamylated heavy chain. It is known in the state of the art that such post-translational modification by N-terminal pyroglutamylation does not have a strong influence on the activity of the antibody (Anal. Biochem., 2006; 348: 24-39).

[030] In one embodiment, the Fab fragment of the human anti-CEACAM5 antibody of the present invention is a Fab fragment of the human anti-CEACAM5 antibody having the following characteristic: a Fab fragment of the human anti-CEACAM5 antibody comprising a chain fragment heavy comprising a variable region of the heavy chain derived from a variable region of the heavy chain consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 by modification of glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid and a light chain comprising a variable region of the light chain consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4.

[031] In another embodiment, the Fab fragment of the human anti-CEACAM5 antibody of the present invention is a Fab fragment of the human anti-CEACAM5 antibody having the following characteristic: a Fab fragment of the human anti-CEACAM5 antibody comprising a chain fragment heavy derived from a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 by modifying the glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[032] The present invention also includes a Fab fragment of the human anti-CEACAM5 antibody having the following characteristic: a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a heavy chain variable region comprising CDR1 consisting of a sequence of amino acids from amino acid positions 31 to 35 of SEQ ID NO: 2, CDR2 consisting of a sequence of amino acids from amino acid positions 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of a sequence of amino acids a from amino acid positions 99 to 110 of SEQ ID NO: 2, and a light chain comprising a variable region of the light chain comprising CDR1 consisting of an amino acid sequence from amino acid positions 24 to 38 of SEQ ID NO: 4, CDR2 consisting of an amino acid sequence from amino acid positions 54 to 60 of SEQ ID NO: 4 and CDR3 consisting of an amino acid sequence from positions d and amino acids 93 to 101 of SEQ ID NO: 4.

[033] The Fab fragment of the human anti-CEACAM5 antibody of the present invention binds to human CEACAM5. A method for measuring the binding activity of the Fab fragment of the human anti-CEACAM5 antibody obtained against human CEACAM5 includes methods such as surface plasmon resonance (SPR) analysis and ELISA. In the case of using, for example, SPR analysis, an association rate constant (ka), a dissociation rate constant (kd), and a dissociation constant (KD) can be measured using Biacore T200 (GE Healthcare Japan Corp.), immobilizing Biotin CAPture Kit (GE Healthcare Japan Corp.) and biotinylated human CEACAM5 on a sensor chip and adding a serially diluted Fab fragment to it.

[034] The Fab fragment of the human anti-CEACAM5 antibody of the present invention can be readily prepared by those skilled in the art using a method known in the art based on sequence information in the heavy chain fragment and the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention disclosed herein. The Fab fragment of the human anti-CEACAM5 antibody of the present invention can be produced according to, but not particularly limited to, a method described in, for example, Method for producing Fab fragment of the human anti-CEACAM5 antibody of the present invention mentioned below .

Conjugate of the present invention

[035] The conjugate of the present invention is a conjugate comprising a labeling portion and the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[036] The "labeling portion" is (i) a binder and a binding agent, (ii) a binder, (iii) a fluorescent dye and a binding agent, or (iv) a fluorescent dye. A particular form is (i) a binder and a binding agent, or (ii) a binder comprising a metal. A certain form is

(iii) a fluorescent dye and a binding agent. A certain form is (i) a binder and a binding agent. The "labeling portion" binder can further comprise a metal. One form is (i) a binder and a bonding agent comprising a metal or (ii) a binder comprising it, and another form is (i) a binder forming a chelate complex with a metal, and a binding agent bond, or (ii) a binder forming a chelate complex with a metal.

[037] The conjugate of the present invention comprising a metal, a fluorescent dye or a non-metallic radioisotope can be used in various contrast media and the like and is used in, for example, a contrast medium for magnetic resonance, a PET tracer , and a fluorescence-labeled molecular imaging agent.

[038] In this specification, "metal" means a paramagnetic metallic ion or a metallic radioisotope. The metal is not particularly limited, as long as the metal forms a coordinated bond with each binder.

A known and appropriate combination of a binder and the metal is selected according to the purpose of use of the conjugate.

[039] The paramagnetic metal ion is properly used in a contrast medium for RMI. Examples of the shape of the paramagnetic metal ion include, but are not limited to, Fe2 +, Fe3 +, Cu2 +, Ni2 +, Rh2 +, Co2 +, Gd3 +, Eu3 +, Dy3 +, Tb3 +, Pm3 +, Nd3 +, Tm3 +, Ce3 +, Y3 +, Ho3 +, Er3 +, La3 +, Er3 +, La3 +, Yb3 +, Mn3 + and Mn2 +. A given shape is Gd3 +, Mn3 +, Mn2 +, Fe2 + or Fe3 +. A given form is Mn3 + or Mn2 +. In this case, halogen or similar can be used as a counter-anion in the conjugate. Alternatively, the counter-anion can be C (= O) O- of the ligand. The conjugate may also contain a counter-cation, such as Na +.

[040] The metal radioisotope is used in, for example, a PET marker. Examples of the shape of the metal radioisotope include, but are not limited to, 89Zr, 51Mn, 52Fe, 60Cu, 67Ga, 68Ga, 72As, 99mTc and 111In. A certain shape is 89Zr, 60Cu, 67Ga, 68Ga, 99mTc or 111In. A certain shape is a zirconium radioisotope. A certain shape is 89Zr.

[041] The "ligand" is a portion capable of forming a chelate complex with a metal in this conjugate and means a group consisting of a chelating agent in a given form. The constituted group is a group that has a bond by removing a proton from the chelating agent.

[042] The "chelating agent" is a compound that can form a coordinated bond with a metal.

[043] Examples of "chelating agent" include siderophore and non-siderophore. Examples of a particular form include MAG3 (mercaptoacetyl-glycyl-glycyl-glycine, CAS No: 66516-09-4) reactive and known derivatives thereof. Examples of siderophore include hydroxamic acid type, catechol type, and mixed ligand type. Examples of hydroxamic acid-like siderophore include ferricrome, deferoxamine (DFO) represented by the following formula:

[044] [Chemical formula 5], fusarinin C, ornibactin, rodotorulic acid and its known reactive derivatives. Examples of catechol-like siderophore include enterobactin, bacillibactin, vibriobactin, and the known reactive derivatives thereof.

Examples of siderophore of the mixed ligand type include azotobactin, pyotrin, yersiniabactin, and their known reactive derivatives. In the case of the siderophore, DFO can react through its reactive functional group -NH2 with the linker or Fab fragment, and the siderophore other than DFO can also react through its reactive functional group, such as a carboxy group, a group hydroxy, or an amino group with the linker or Fab fragment by a method normally used by those skilled in the art.

[045] Examples of non-siderophore include DTPA (diethylene triamine pentaacetic acid, CAS No: 67-43-6), DTPA-BMA (1,7-bis (methylcarbamoylmethyl) -1,4,7-triazaheptane- 1,4,7-triacetic, CAS No: 119895-95-3), EOB-DTPA (ethoxybenzyl-DTPA, 2 - [[(2S) -2- [bis (carboxymethyl) amino] -3- (4- ethoxyphenyl) propyl] - [2- [bis (carboxymethyl) amino] ethyl] amino] acetic), TTHA (hexa-acetic triethylene tetramino acid, CAS No: 869-52-3), DO3A (acid 1,4,7, 10- tetra-azacyclododecane-1,4,7-triacetic, CAS No: 217973-03-0), HP-DO3A (10- (2-hydroxypropyl) -1,4,7,10-tetra-azacyclododecane-1 acid , 4,7-triacetic, CAS No: 120041-08-9), DOTA (1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid, CAS No: 60239-18 -1) and its known reactive derivatives.

[046] The compounds and conjugates described in this document also include free forms and their salts, unless otherwise specified.

In this context, the "salt thereof" is a salt that can be formed by the compound or the conjugate that can form an acid addition salt or a salt with a base, depending on the type of a substituent on the compound or the conjugate.

Specific examples thereof include: acid addition salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid, or organic acids, such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltestartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid aspartic acid, and glutamic acid; salts with inorganic bases, such as sodium, potassium, magnesium, calcium and aluminum, or organic bases, such as methylamine, ethylamine, ethanolamine, lysine and ornithine; salts with various amino acids and derivatives of amino acids, such as acetileukin; and ammonium salts. For example, DFO exists as deferoxamine methanesulfonate or exists as other salts. DTPA exists both in free form and as sodium salt.

[047] A certain form of the "chelating agent" for use in a contrast medium for RMI is the siderophore or non-siderophore chelating agent described above.

[048] A certain form of the "chelating agent" for use in a PET tracer is the siderophore or non-siderophore chelating agent described above.

A certain shape is MAG3. One form is DFO.

[049] Examples of a particular form of the "chelating agent" that constitutes the linker contained in the conjugate of the present invention include DFO, DTPA, DTPA-BMA, EOB-DTPA, DO3A, HP-DO3A and DOTA. A certain form is DFO, DTPA or DOTA. One form is DFO.

[050] The "linker" is a group that creates a distance between the Fab fragment of the human anti-CEACAM5 antibody and the linker. Examples of a particular form of the "linker" in the conjugate include the following formula:

[051] [Chemical formula 6] (hereinafter referred to as C (= S) -NH- (1,4-phenylene) -NH-C (= S) -), -CH2- (1,4-phenylene) - NH-C (= S) -, and -C (= O) - (C1-20 alkylene) -C (= O) -. In this context, "C1-20 alkylene" is straight or branched alkylene with 1 to 20 carbon atoms. A certain form of C1-20 alkylene is C1-10 alkylene or C1-2 alkylene. A certain form of C1-20 alkylene is ethylene. A certain form is C (= S) -NH- (1,4-phenylene) -NH-C (= S) -. A given form is -C (= O) -C2H4- C (= O) -. Examples of a reagent that can be used to form the binding agent include HO-C (= O) - (C1-20 alkylene) -C (= O) -OH, succinic acid and p-phenylene diisothiocyanate.

[052] The conjugate of the present invention comprising a fluorescent dye can be used as a fluorescently labeled molecular imaging agent.

[053] A dye with a maximum absorption and a maximum emission at the near infrared wavelength (650 to 1000 nm) normally used in the photoimage can be used as a fluorescent dye to be used in the conjugate of the present invention. Examples of a particular form of the fluorescent dye include cyanine and indocyanine compounds.

Examples of a particular form include IRDye800CW (LI-COR, Inc.), Cy (Molecular Probes, Inc.), Alexa Fluor, BODIPY and DyLight (Thermo Fisher Scientific Inc.), CF790 (Biotium, Inc.), DY (Dyomics GmbH), HiLyte Fluor 680 and HiLyte Fluor 750 (AnaSpec Inc.) and PULSAR650 and QUASAR670 (LGC Biosearch Technologies). One form is the IRDye800CW. The fluorescent dye can be reacted through its carboxy group, hydroxy group, amino group or the like or through an active group introduced by a method commonly used by those skilled in the art with the Fab fragment or the binding agent. A certain form of the fluorescent dye that has an active group introduced is a fluorescent dye esterified with an N-hydroxysuccinimide (NHS) group. For example, the NHS esters of IRDye800CW mentioned above are commercially available and can be used.

[054] The conjugate of the present invention comprising a non-metallic radioisotope can be used as a PET tracer.

[055] Examples of the non-metallic radioisotope for use in the conjugate of the present invention include 18F, 11C, 15O and 13N. A certain shape is 18F.

For example, N-succinimidyl-4- [18F] fluorobenzoic acid ([18F] SFB) can be used for the attachment of a compound comprising the non-metallic radioisotope.

[056] The binding of the Fab fragment of the human anti-CEACAM5 antibody of the present invention to the labeling portion can be suitably performed by those skilled in the art using a known approach. For example, the labeling moiety can be linked to one or more amino groups (for example, an N-terminal amino group and an amino group of an amino acid side chain), to one or more thiol groups (for example, a thiol group thiol of an amino acid side chain) or one or more carboxyl groups (e.g., C-terminal carboxyl groups and an amino acid side chain) of the Fab fragment of the human anti-CEACAM5 antibody of the present invention. A particular form of the conjugate of the present invention is a conjugate in which the labeling moiety is linked to one or more amino groups of the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[057] When the labeling portion is a ligand and a binding agent, the conjugate of the present invention can be produced by reacting the chelating agent that forms the ligand with a substance obtained by reacting the Fab fragment of the human anti-CEACAM5 antibody of the present invention with the binding agent. It can be produced by reacting the Fab fragment of the human anti-CEACAM5 antibody of the present invention with a substance obtained by reacting the chelating agent that forms the ligand with the binding agent. As a reaction example, a substance obtained by reacting the amino group of the chelating agent with the linker reacts with one or more amino groups (for example, an N-terminal amino group and an amino group on a lysine side chain) of Fab fragment of the human anti-CEACAM5 antibody of the present invention. When the labeling portion is a linker, it can be produced by reacting the chelating agent that forms the linker with the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

As an example of reaction, the chelating agent reacts with one or more amino groups (e.g., an N-terminal amino group and an amino group on a lysine side chain) of the Fab fragment of the human anti-CEACAM5 antibody of the present invention. The reaction of synthesizing thiourea by adding isothiocyanate to the amine, the reaction of synthesizing amide by adding carboxylic acid to the amine or the like, can be used in the production of the conjugate of the present invention.

The reaction can be carried out by applying a method known to those skilled in the art. A binder compound previously bound to the binding agent can be used as a starting material. Examples of the binder compound bound to the binding agent include p-SCN-Bn-DFO (DFO replaced by a p-isothiocyanophenylaminothiocarbonyl group, CAS number: 1222468-90-7) represented by the following formula:

[058] [Chemical formula 7]

OH O H N HN N N H S O SCN OH O

H H3 C N N

N

OO OH, DTPA replaced by a p-isothiocyanobenzyl group (p-SCN-Bn-DTPA, CAS No .: 102650-30-6), DOTA replaced by a p-isothiocyanobenzyl group (p-SCN- DOTA-Bn, CAS No .: 127985-74-4) and p-SCN-Bn-CHX-A "-DTPA acid ([(R) -2-amino-3- (4-isothiocyanatophenyl) propyl] -trans- (S, S) - cyclohexane-1,2-diamine-penta-acetic, CAS No .: 157380-45-5).

[059] The conjugate of the present invention can be produced as, for example, a conjugate comprising the Fab fragment attached to at least a labeling portion through amino groups by the reaction of one or more amino groups (for example, an N amino group -terminal and an amino group of an amino acid side chain) of the Fab fragment of the human anti-CEACAM5 antibody of the present invention with the labeling portion having an isocyanic acid group.

[060] The metal (paramagnetic metal ion or metal radioisotope) can be added to the Fab fragment of the human anti-CEACAM5 antibody of the present invention attached to at least a portion of the tag, then produced by the production method, in order to obtain the conjugate of the present invention comprising the metal.

[061] The conjugate of the present invention is a conjugate comprising the Fab fragment of the human anti-CEACAM5 antibody of the present invention attached to at least a tag portion. A particular form of the conjugate of the present invention is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 25 tag portions. A certain form is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 23 labeling portions. A particular form is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 16 tag portions.

One form is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 11 labeling portions. One particular form is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 10 labeling portions.

One particular form is the Fab fragment of the human anti-CEACAM5 antibody linked to 1 to 9 labeling portions. A particular form is the Fab fragment of the human anti-CEACAM5 antibody linked to 4 to 23 labeled portions. A certain form is the Fab fragment of the human anti-CEACAM5 antibody linked to 4 to 16 labeling portions. A certain form is the Fab fragment of the human anti-CEACAM5 antibody linked to 4 to 10 labeling portions.

One particular form is the Fab fragment of the human anti-CEACAM5 antibody linked to 4 to 9 tag portions. One form is the Fab fragment of the human anti-CEACAM5 antibody linked to 3 to 23 labeled portions. A certain form is the Fab fragment of the human anti-CEACAM5 antibody linked to 3 to 16 labeling portions. One form is the Fab fragment of the human anti-CEACAM5 antibody linked to 3 to 10 labeling portions.

One form is the Fab fragment of the human anti-CEACAM5 antibody linked to 3 to 9 labeling portions. A particular form of the conjugate of the present invention is the Fab fragment of the human anti-CEACAM5 antibody attached to at least a labeling portion further comprising a metal.

A particular form of the conjugate of the present invention can be a mixture of conjugates that differ in the number of linked label moieties.

[062] In one embodiment, the conjugate of the present invention is a conjugate comprising a labeling portion and the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[063] In a given embodiment, the conjugate of the present invention is a conjugate in which the labeling moiety is (i) a linker and a linker, or (ii) a linker.

[064] In a given embodiment, the conjugate of the present invention is a conjugate, wherein the labeling portion is (i) a linker and a linker, or (ii) a linker, and the linker is a linker represented by the following formula (A):

[065] [Chemical formula 8]

(A) where the wavy line represents the binding to the Fab fragment of the human anti-CEACAM5 antibody or to the binding agent.

[066] In a given embodiment, the conjugate of the present invention is a conjugate, where the labeling portion is (i) a linker and a linker, and the linker and the linker are a group represented by following formula (A '):

[067] [Chemical formula 9] (A ') where the wavy line represents the binding to the Fab fragment of the human anti-CEACAM5 antibody.

[068] In a given embodiment, the conjugate of the present invention is a conjugate, where the labeling portion is a group represented by the formula (A '), and the Fab fragment of the human anti-CEACAM5 antibody is attached to the atom carbon of a terminal C (= S) group of the labeling portion through an amino group on the Fab fragment.

[069] In a given embodiment, the conjugate of the present invention is a conjugate represented by the following formula (I): (LX) p-Ab (I) where Ab is the Fab fragment of the human anti-CEACAM5 antibody of the present invention; L is the ligand; X is the bonding agent or a bond; and p is a natural number from 1 to 25.

[070] A given shape of p is a natural number from 1 to 23. A given shape is a natural number from 1 to 16. A given shape is a natural number from 1 to 11. A given shape is a natural number from 1 to 10. A given shape is a natural number from 1 to 9. A given shape is a natural number from 4 to 23. A given shape is a natural number from 4 to 16. A given shape is a natural number from 4 to 10 A given shape is a natural number from 4 to 9. A given shape is a natural number from 3 to 23. A given shape is a natural number from 3 to

16. A given shape is a natural number from 3 to 10. A given shape is a natural number from 3 to 9.

[071] In a given embodiment, the conjugate of the present invention is a conjugate of formula (I), where L is a linker represented by the following formula (A):

[072] [Chemical formula 10]

(A) where the wavy line represents the bond to X (or Ab when X is a bond).

[073] In a given embodiment, the conjugate of formula (I) is a conjugate represented by the following formula (II):

[074] [Chemical formula 11] (II) p where Ab is the Fab fragment of the human anti-CEACAM5 antibody of the present invention; and p is a natural number from 1 to 25.

[075] A given shape of p is a natural number from 1 to 23. A given shape is a natural number from 1 to 16. A given shape is a natural number from 1 to 11. A given shape is a natural number from 1 to 10. A given shape is a natural number from 1 to 9. A given shape is a natural number from 4 to 23. A given shape is a natural number from 4 to 16. A given shape is a natural number from 4 to 10 A given shape is a natural number from 4 to 9. A given shape is a natural number from 3 to 23. A given shape is a natural number from 3 to

16. A given shape is a natural number from 3 to 10. A given shape is a natural number from 3 to 9.

Ab is attached to the carbon atom of the terminal group C (= S) of the labeling portion through an amino group on Ab.

[076] In a given embodiment, the conjugate of the present invention is a conjugate of formula (I) which further comprises a metal. A certain shape of the metal is a metallic radioisotope. A certain form of the radioisotope metal is 89Zr.

[077] In a given embodiment, the conjugate of the present invention is a conjugate of formula (II) which further comprises a metal. A certain shape of the metal is a metallic radioisotope. A certain form of the radioisotope metal is 89Zr.

[078] The conjugate of the present invention also includes a conjugate that is a mixture of a plurality of conjugates. For example, a conjugate, which is a mixture of a conjugate comprising a tag portion and a Fab fragment of the post-translationally unmodified human anti-CEACAM5 antibody of the present invention and a conjugate comprising a tag portion and the Fab fragment of the antibody anti-human CEACAM5 of the present invention resulting from post-translational modification of the Fab fragment of the human anti-CEACAM5 antibody, is also included in the conjugate of the present invention.

[079] Certain embodiments of the conjugate of the present invention, which is a mixture of a plurality of conjugates of the present invention, will be shown below: (1) A conjugate, which is a mixture of a conjugate, in which the labeling portion is a binder and a binding agent represented by the formula (A '),

and the Fab fragment of the human anti-CEACAM5 antibody is a Fab fragment of the human anti-CEACAM5 antibody which comprises a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4, and a conjugate, wherein the labeling portion is a linker and binding agent represented by the formula (A '), and the Fab fragment of the human anti-CEACAM5 antibody is a Fab fragment of the antibody human anti-CEACAM5 comprising a heavy chain fragment derived from a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 by modifying glutamine at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid, and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

(2) The conjugate of (1), further comprising a metal.

(3) A conjugate that is a mixture of a conjugate of (1) further comprising a metal and a conjugate of (1) not comprising metal.

(4) The conjugate of (2) or (3), in which the metal is a metallic radioisotope.

(5) The conjugate of (4), in which the metal radioisotope is 89Zr.

Polynucleotide of the present invention

[080] The polynucleotide of the present invention includes a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the anti-human CEACAM5 antibody of the present invention.

[081] In one embodiment, the polynucleotide of the present invention is a polynucleotide containing a nucleotide sequence that encodes a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of the SEQ ID NO: 2, or a polynucleotide containing a nucleotide sequence that encodes a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4.

[082] Examples of the polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2, include a polynucleotide containing a nucleotide sequence from nucleotide positions 1 to 363 of SEQ ID NO: 1.

[083] Examples of the polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4, include a polynucleotide containing a nucleotide sequence from nucleotide positions 1 to 336 of SEQ ID NO: 3.

[084] In a preferred embodiment, the polynucleotide of the present invention is a polynucleotide containing a nucleotide sequence that encodes a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2, or a polynucleotide containing a nucleotide sequence which encodes a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[085] Examples of the polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2, include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1.

[086] Examples of the polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4 include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3.

[087] The polynucleotide of the present invention is synthesizable by the use of a genetic synthesis method known in the art based on the nucleotide sequence delineated from the amino acid sequences of the heavy chain fragment and the light chain of the anti antibody Fab fragment - human ECACAM5 of the present invention. Various methods known to those skilled in the art, such as methods for the synthesis of an antibody gene described in International Publication No. WO 90/0786 1, can be used as methods of genetic synthesis.

Expression vector of the present invention

[088] The expression vector of the present invention includes an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention, an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[089] The expression vector of the present invention preferably includes an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2, an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4, and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2 and one polynucleotide containing a nucleotide sequence that encodes a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4.

[090] The expression vector of the present invention preferably includes an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2, an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4, and an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a heavy chain fragment consisting of the represented amino acid sequence by SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

[091] The expression vector of the present invention is not particularly limited as long as a polypeptide encoded by the polynucleotide of the present invention can be produced in various host cells of prokaryotic cells and / or eukaryotic cells. Examples of this expression vector include plasmid vectors and viral vectors (for example, adenovirus and retrovirus). It is preferable to use pEE6.4 or pEE12.4 (Lonza Group AG).

[092] The expression vector of the present invention may comprise a promoter operably linked to a gene encoding the heavy and / or light chain fragment in the polynucleotide of the present invention. Examples of the promoter to express the Fab fragment of the present invention in a host cell include the Trp promoter, the lac promoter, the recA promoter, the λPL promoter, the lpp promoter and the tac promoter when the host cell is a bacterium of the Escherichia genus. Examples of the promoter for expression in yeast include the PH05 promoter, the PGK promoter, the GAP promoter and the ADH promoter. Examples of the promoter for expression in bacteria of the genus Bacillus include the SL01 promoter, the SP02 promoter and the penP promoter.

Examples of them include virus-derived promoters such as CMV, RSV and SV40, retrovirus promoter, actin promoter, EF (elongation factor) 1α promoter and heat shock promoter when the host is a eukaryotic cell, such as a mammalian cell .

[093] In the case where a bacterium is used, particularly E. coli, as a host cell, the expression vector of the present invention may further comprise a start codon, a stop codon, a termination region and a replicable unit. On the other hand, in the case where a yeast, an animal cell or an insect cell is used as a host, the expression vector of the present invention can comprise a start codon and a stop codon. In this case, an enhancer or enhancer sequence, 5 'and 3' untranslated regions of a gene encoding the heavy and / or light chain fragment of the present invention, a secretion signal sequence, a splicing junction, a polyadenylation site or a replicable unit etc. may be contained therein. In addition, a normally used selective marker (eg, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene and dihydrofolate reductase gene) may be contained therein according to goal.

Transformed host cell of the present invention

[094] The transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention, selected from the group consisting of the following items (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody of the present invention; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention;

(c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain the Fab fragment of the human anti-CEACAM5 antibody of the present invention; and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention and an expression vector comprising a polynucleotide containing a sequence nucleotide encoding the Fab chain of the human anti-CEACAM5 antibody Fab fragment of the present invention.

[095] In one embodiment, the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention, selected from the group consisting of the following items (a) to (d): (a ) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4;

(c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4; and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2 and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4.

[096] In one embodiment, the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention, selected from the group consisting of the following items (a) to (d): (a ) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2;

(b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4; (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding lightweight consisting of the amino acid sequence represented by SEQ ID NO: 4; and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[097] Preferred examples of the transformed host cell of the present invention include a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention, and a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the fragment's heavy chain fragment Fab of the human anti-CEACAM5 antibody of the present invention and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[098] The host cell to be transformed is not particularly limited, as long as it is compatible with the expression vector used and can be transformed with the expression vector to express the Fab fragment.

Examples of these include various cells, such as natural cells and artificially established cells, generally used in the technical field of the present invention (eg bacteria (bacteria of the genus Escherichia and bacteria of the genus Bacillus), yeasts (the genus Saccharomyces, the genus Pichia, etc.), animal cells and insect cells (for example, Sf9)) and mammalian cell lines (for example, cultured cells, such as CHOK1SV cells, CHO-DG44 and 293 cells). The transformation itself can be carried out by a known method, for example, a calcium phosphate method or an electroporation method.

Method for producing the Fab fragment of the human anti-CEACAM5 antibody according to the present invention

[099] The method for producing a Fab fragment of the human anti-CEACAM5 antibody according to the present invention comprises the step of culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody.

[0100] In one embodiment, the transformed host cell of the present invention to be cultured in the method for producing a Fab fragment of the human anti-CEACAM5 antibody according to the present invention is selected from the group consisting of the following items (a) to (c):

(a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain the Fab fragment of the human anti-CEACAM5 antibody of the present invention; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment of the present invention and an expression vector comprising a polynucleotide containing a nucleotide sequence which encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention; and one (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody of the present invention and a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[0101] A particular form of the transformed host cell of the present invention to be cultured in the method for producing a Fab fragment of the human anti-CEACAM5 antibody according to the present invention is selected from the group consisting of the following items (a ) to (c): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by the amino acid positions 1 to 121 of SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2 and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4; and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment comprising a variable region of the heavy chain consisting of the amino acid sequence represented by amino acid positions 1 to 121 of SEQ ID NO: 2, and a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a light chain comprising a variable region of the light chain consisting of the amino acid sequence represented by amino acid positions 1 to 112 of SEQ ID NO: 4.

[0102] A certain form of the transformed host cell of the present invention to be cultured in the method for producing a fragment

Fab of the human anti-CEACAM5 antibody according to the present invention is selected from the group consisting of the following items (a) to (c): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a sequence nucleotide encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and an expression vector comprising a polynucleotide containing a sequence nucleotide encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4; and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[0103] The transformed host cell of the present invention used is preferably a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention or a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence which encodes the Fab chain fragment of the Fab fragment human anti-CEACAM5 antibody of the present invention and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody of the present invention.

[0104] In the method for producing a Fab fragment of the human anti-CEACAM5 antibody according to the present invention, the transformed host cell can be grown in a nutrient medium. The nutrient medium preferably contains a carbon source, a source of inorganic nitrogen or a source of organic nitrogen necessary for the growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch and sucrose. Examples of the source of inorganic nitrogen or the source of organic nitrogen include ammonium salts, nitrates, amino acids, millhocin, peptone, casein, meat extracts, soy bran and potato extracts. In addition, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate and magnesium chloride), vitamins and antibiotics (for example, tetracycline, neomycin, ampicillin and kanamycin)) may be contained in the even if desired.

[0105] The culture of the transformed host cell itself is performed by a known method. The culture conditions, for example, temperature, average pH and culture time, are appropriately selected. When the host is, for example, an animal cell, MEM medium (Science; 1952; 122: 501), DMEM medium (Virology; 1959; 8: 396-97), RPMI1640 medium (J. Am. Med.

Assoc.; 1967; 199: 519-24), medium 199 (Proc. Soc. Exp. Biol. Med .; 1950; 73: 1-

8), or the like containing approximately 5 to 20% fetal bovine serum can be used as a medium. Preferably, the average pH is approximately 6 to

8. Culture is normally carried out at approximately 30 to 40 ° C for approximately 15 to 336 hours, and aeration or agitation can also be carried out, if necessary. When the host is an insect cell, its examples include Grace's medium (PNAS; 1985; 82: 8404-8) containing fetal bovine serum. Preferably, its pH is approximately 5 to 8. Culture is normally carried out at approximately 20 to 40 ° C for 15 to 100 hours, and aeration or agitation can also be carried out, if necessary. When the host is a bacterium, an actinomycete, a yeast or a filamentous fungus, for example, a liquid medium containing the nutrient source described above is appropriate. A medium with pH 5 to 8 is preferred. When the host is E. coli, preferred examples of medium include LB medium and M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory; 1972: 431).

In this case, the culture can generally be carried out at 14 to 43 ° C for approximately 3 to 24 hours with aeration or agitation, if necessary. When the host is a bacterium of the genus Bacillus, it can generally be carried out between 30 and 40 ° C for approximately 16 to 96 hours with aeration or agitation, if necessary. When the host is a yeast, examples of the medium include the minimum Burkholder medium (PNAS; 1980; 77: 4505-8). The desired pH is between 5 and 8. The culture is usually carried out at approximately 20 to 35 ° C for approximately 14 to 144 hours, and aeration or agitation can also be carried out, if necessary.

[0106] The method for producing a Fab fragment of the human anti-CEACAM5 antibody according to the present invention may comprise the step of recovering, preferably isolating or purifying, the Fab fragment of the expressed human anti-CEACAM5 antibody, in addition to the step of carrying out culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody. Examples of the isolation or purification method include: methods that explore solubility, such as the salting out and solvent precipitation method; methods that explore the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis; charge-exploiting methods, such as ion exchange chromatography and hydroxylapatite chromatography; methods that explore specific affinity, such as affinity chromatography; methods that explore differences in hydrophobicity, such as high performance liquid chromatography and reverse phase; and methods that explore the difference in the isoelectric point, such as isoelectric focus.

Method for the production of the conjugate according to the present invention

[0107] The method for producing a conjugate according to the present invention comprises the step of covalently linking the Fab fragment of the human anti-CEACAM5 antibody of the present invention to a labeling portion. The method for producing a conjugate according to the present invention can also comprise the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; and covalently attaching the Fab fragment to a labeling portion. The method for producing a conjugate according to the present invention can also comprise the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; recovering the expressed Fab fragment; and covalently attaching the Fab fragment to a labeling portion. The binding agent, binder or fluorescent dye,

etc., and the method of attachment used can use those described in the Conjugate of the present invention.

[0108] In one embodiment, the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the anti-antibody Fab fragment - human CEACAM5; and covalently attaching the Fab fragment to a labeling portion. A particular form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; recovering the expressed Fab fragment; and covalently attaching the Fab fragment to a labeling portion.

[0109] A certain form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody ; and i) attaching the Fab fragment via a linker to a linker or ii) covalently attaching the Fab fragment directly to a linker. A particular form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; recovering the expressed Fab fragment; and i) attaching the Fab fragment via a linker to a linker or ii) covalently attaching the Fab fragment directly to a linker.

[0110] A particular form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody ; covalently attach the Fab fragment via a linker or directly to a linker; and attaching a metal to the conjugate linker through a coordinated bond (i.e., forming a chelate complex). A particular form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; recovering the expressed Fab fragment; and covalently attaching the Fab fragment via a linker or directly to a linker; and connecting a metal to the conjugate linker through a coordinated connection.

[0111] A certain form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody ; and covalently attaching the Fab fragment via a linker or directly to a linker; and attaching a metal radioisotope to the conjugate ligand through a coordinated bond (i.e., forming a chelate complex). A particular form of the method for producing a conjugate according to the present invention is a method comprising the steps of: culturing the transformed host cell of the present invention to express the Fab fragment of the human anti-CEACAM5 antibody; recovering the expressed Fab fragment; and covalently attaching the Fab fragment via a linker or directly to a linker; and attaching a metal radioisotope to the conjugate linker via a coordinated link.

[0112] The method for producing a conjugate according to the present invention can be carried out as a method comprising two or more of the steps defined above as a series of steps or can be carried out as a method comprising at least one of the steps defined above. For example, a method comprising the step of attaching the Fab fragment of the human anti-CEACAM5 antibody of the present invention to a labeling portion and a method comprising the step of labeling the Fab fragment of the human anti-CEACAM5 antibody of the present invention attached to the portion metal marking are also included in the method for producing a conjugate according to the present invention. In addition, the method for producing a conjugate according to the present invention includes a method with a different order of steps. For example, a method comprising labeling a linker with a metal radioisotope and then covalently attaching the linker to the Fab fragment of the human anti-CEACAM5 antibody of the present invention are also included in the method for producing a conjugate according to the present invention.

Diagnostic composition and diagnostic method of the present invention

[0113] The present invention relates to a diagnostic composition comprising the conjugate of the present invention comprising a fluorescent dye, a metallic or non-metallic radioisotope (hereinafter, referred to as the detectable conjugate of the present invention). The detectable conjugate of the present invention can be formulated according to a routine method and used as a drug for clinical staging (particularly, for the diagnosis of cancer). The drug for clinical staging means a diagnostic drug capable of examining the degree of progression of a medical condition. For example, for cancers, it means a diagnostic drug capable of examining their clinical stage. The cancer that can be diagnosed by the diagnostic composition of the present invention is selected from the group consisting of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland and a cancer resulting from their metastases. Cancer is preferably colorectal cancer or cancer resulting from colorectal cancer metastasis. Cancer is most preferably a cancer resulting from colorectal cancer metastasis. This type of cancer includes liver metastasis.

[0114] The amount of the detectable conjugate of the present invention added to the formulation of the diagnostic composition of the present invention differs depending on the degree of symptoms or age of a patient, the dosage form of a preparation used or the binding title of the Fab fragment, etc. For example, approximately 0.001 mg / kg to 100 mg / kg based on the mass of the Fab fragment can be used per unit of body weight of a patient.

[0115] Examples of the dosage form of the diagnostic composition of the present invention can include parenteral agents, such as injections and drip infusion agents. Administration is preferably performed by intravenous injection, local intramuscular injection into a target, subcutaneous injection or the like. For the formulation, a carrier or additive suitable for these dosage forms can be used in a pharmaceutically acceptable range. The type of pharmaceutically acceptable vehicle or additive is not particularly limited, and a carrier or additive well known to those skilled in the art can be used.

[0116] The present invention also relates to the use of the detectable conjugate of the present invention for the production of a composition for diagnosing cancer or a composition for staging in a given form. The present invention also relates to the detectable conjugate of the present invention for use in the diagnosis of cancer or for use in staging in a particular way.

[0117] In addition, the present invention also relates to a method for the diagnosis of a cancer, which comprises administering pre- or intraoperatively the detectable conjugate of the present invention to an individual.

In this context, the "individual" is a human or any other mammal that needs to be diagnosed. A certain form is a human being who needs to receive the diagnosis. The effective amount of the detectable conjugate of the present invention in the diagnostic method of the present invention can be the same amount as the effective amount of the detectable conjugate of the present invention for the formulation described above. In the diagnostic method of the present invention, the detectable conjugate of the present invention is preferably administered by intravenous injection or the like. The conjugate of the present invention for use as a PET tracer can be photographed by PET after 1.5 to 48 hours, after 2 to 48 hours in a certain way, after 4 to 24 hours in a certain way, after 4 to 6 hours in a given form or after 1.5 to 6 hours in a given form of administration. In the diagnostic method of the present invention, if a fluorescence-labeled conjugate of the present invention is used in intraoperative diagnosis, the conjugate is administered to a patient, for example, 2 to 48 hours, preferably 4 hours, before the operation.

[0118] In an alternative embodiment, the present invention also relates to the use of the Fab fragment of the human anti-CEACAM5 antibody of the present invention for the production of the conjugate of the present invention. In a given embodiment, the present invention also relates to the use of the Fab fragment of the human anti-CEACAM5 antibody of the present invention for the production of a diagnostic composition comprising the conjugate of the present invention.

[0119] The present invention is described, in general, above. Particular examples will be provided here for reference, in order to gain further understanding. However, these are given for illustrative purposes only and do not limit the present invention.

EXAMPLES Example 1: Preparation of the Fab fragment of the human anti-CEACAM5 antibody

[0120] An antibody with variable regions that does not attenuate affinity, even by binding a labeling portion, was developed using a molecular model of a humanized antibody constructed according to the literature (Proteins: Structure, Function, and Bioinformatics; 2014 ; 82: 1624-1635) after humanization of the T84.66 mouse-derived human anti-CEACAM5 antibody with reference to the method described in the literature (Protein Eng. Des. Sel .; 2004; 17: 481-489).

[0121] A gene encoding a signal sequence (Protein Engineering; 1987; 1: 499-505) and a gene from the human Igγ1 Fab region (consisting of a nucleotide sequence of nucleotide positions 364 to 678 of SEQ ID NO : 1) were linked to the 5 'and 3' side, respectively, of the antibody heavy chain variable region gene, and this heavy chain fragment gene was inserted into the GS pEE6.4 vector (Lonza Group AG). In addition, a gene encoding a signal sequence and a gene for the human Igκ constant region (consisting of a nucleotide sequence of nucleotide positions 337 to 657 of SEQ ID NO: 3) were connected to the 5 'side and to the side 3 ', respectively, of the variable region gene of the antibody light chain, and that light chain gene was inserted into the GS pEE12.4 vector (Lonza Group AG). The pEE vectors mentioned above, having the insertions of the light chain genes and the antibody heavy chain fragment, respectively, were cleaved with the restriction enzymes NotI and PvuI and ligated using the TAKARA Ligation Kit Vide 2.1 ligation kit (Takara Bio Inc.) to construct a GS vector having both light chain and heavy chain fragment insertions.

[0122] The antibody was expressed by two types of methods, transient and constitutive expression, using the GS vector mentioned above, having both light chain and heavy chain fragment insertions. For transient expression, Expi293F cells (Thermo Fisher Scientific Inc.) grown in approximately 3000000 cells / mL in Expi293 expression medium (Thermo Fisher Scientific Inc.) were transfected with the GS vector mentioned above having the light chain gene inserts and the heavy chain fragment using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc.) and cultured for 5 to 7 days. The culture supernatant was purified using KappaSelect (GE Healthcare Japan Corp.) to obtain a Fab fragment. For constitutive expression, CHOK1SV cells (Lonza Group AG) were transfected with a linear vector obtained with PvuI from the GS vector mentioned earlier , with insertions of the light chain genes and the heavy chain fragment, by electroporation using Gene Pulser (Bio-Rad Laboratories, Inc.). The day after transfection, methionine sulfoximine was added to it, followed by culture for 5 to 7 days. The cells were inoculated in a semi-solid medium containing methyl cellulose. After colony formation, cells with a high level of Fab fragment expression were obtained using ClonePix FL (Molecular Devices, LLC). The cell culture supernatant was purified using Capto L (GE Healthcare Japan Corp.),

Q Sepharose Fast Flow (GE Healthcare Japan Corp.) and BioPro S75 (YMC Co., Ltd.) to obtain a Fab fragment.

[0123] The nucleotide sequence encoding the heavy chain fragment of the prepared human anti-CEACAM5 antibody Fab fragment (designated as PB009-01) is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2. The nucleotide sequence encoding the PB009-01 light chain is shown in SEQ ID NO: 3, and the amino acid sequence encoded by it is shown in SEQ ID NO: 4. The variable region of the heavy chain PB009-01 consists of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 and heavy chain CDR1, CDR2 and CDR3 consist of the amino acid sequences from amino acid positions 31 to 35, 50 to 66 and 99 to 110, respectively, of SEQ ID NO: 2. The PB009-0 light chain variable region consists of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4, and the chains CDR1, CDR2 and CDR3 of the light chain consist of the amino acid sequences of the amino acid positions 24 to 38, 54 to 60 and 93 to 101, respectively, of SEQ ID NO: 4.

[0124] Variable regions and CDR sequences were determined according to Kabat numbering (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda).

Example 2: Production of the Fab fragment conjugate

[0125] p-SCN-Bn-DFO (DFO replaced by a p-isothiocyanophenylaminothiocarbonyl group) (Macrocyclics, Inc.) was used to bind the DFO chelating agent to the Fab fragment PB009-01. A 1/5 amount of a 0.1 M sodium carbonate solution (pH 9.0) was added to a solution of the Fab fragment adjusted to 1 mg / mL with phosphate buffered saline (pH

7.4). p-SCN-Bn-DFO at a final concentration of 1 mg / mL was added and reacted at 37 ° C for 1.5 hours. After the reaction, a Fab fragment bound to DFO via a binding agent (-C (= S) -NH- (1,4-phenylene) -NH-C (= S) -) (designated as PB009-02) was purified using a centrifuge filter - Amicon Ultra 3K-0.5 mL (Merck Millipore).

[0126] The number of binders made up of DFO linked to PB009-02 has been confirmed by mass spectrometry. PB009-02 was desalted using the MassPREP Micro Desalination Column (Waters Corp.) and the measurement was performed using the SYNAPT G2 mass spectrometer (Waters Corp.). As a result, a molecule was confirmed in which at least 3 to 10 DFO linkers were linked to a PB009-01.

Example 3: Assessment of liaison activity

[0127] To measure the detailed binding activity of PB009-02, the analysis was performed by SPR. In this example, an M5A Fab fragment (referred to as M5A-Fab), a humanized antibody from T84.66, was used as a comparative Fab fragment. M5A-Fab linked to DFO via a linker (referred to as M5A-Fab-DFO) was prepared using the method described in Example 2.

[0128] SPR analysis was performed using Biacore T200 (GE Healthcare Japan Corp.). 10 µg / mL of human CEACAM5 (R&D Systems, Inc.) biotinylated with Biotin CAPture Kit, S Series (GE Healthcare Japan Corp.) and Biotin Labeling Kit-NH2 (Dojindo Laboratories) were added to the sensor chip surface at a rate flow rate of 5 µL / min for 2 minutes and immobilized on it. PB009-01, PB009-02, M5A-Fab and M5A-Fab-DFO were diluted with HBS-EP + solution (GE Healthcare Japan Corp.) in 6 series at a rate of 2 times 400 nM, and each 100 μL was added to the channel at a flow rate of 50 µL / min. In this measurement system, the dissociation constant (KD) of

PB009-01, PB009-02, M5A-Fab or M5A-Fab-DFO for human CEACAM5 was calculated using data analysis software (BIA Evaluation, GE Healthcare Japan Corp.). As a result, as shown in the table below, the binding activity of M5A-Fab has been attenuated by the binding of DFO, while the binding activity of PB009-01 has not been attenuated by binding of DFO.

TABLE 1 Fab KD (M) Fab linked to DFO KD (M) PB009-01 1.7E-10 PB009-02 6.1E-10 M5A-Fab 1.6E-10 M5A-Fab-DFO 9,6E-09 Example 4: 89Zr labeling of the Fab fragment conjugate bound to

DFO

[0129] 89Zr was purchased as Zr-Oxalate from 3D Imaging LLC. 20 μL of Zr-oxalate (2.6 mCi) was neutralized with 10 μL of 2 M sodium carbonate. Then, 20 μL of 5 mg / mL of gentisic acid were added to them. In addition, 110 μL of 20 mM HEPES (4- (2-hydroxyethyl) -1- piperazinoethanesulfonic acid) containing 0.05% polysorbate 80 was added to them. 40 μL of 10 mg / mL of PB009-02 was added to it and reacted at room temperature for 30 minutes and then reacted at 37 ° C for 30 minutes. After the reaction, PB009-02 labeled with 89Zr (designated as PB009-03) was purified using a centrifuge filter - Amicon Ultra 3K-0.5 mL (Merck Millipore).

Example 5: Conjugate contrast assessment in the subcutaneous xenograft model

[0130] 1 x 106 cells of the LS174T human colorectal cancer cell line (ATCC (R); CL-188) were xenografted subcutaneously as CEACAM5 positive human cells in the right shoulder of an immunodeficient mouse (NOG; Taconic Biosciences) and 5 x 106 cells of the HCT-15 human colorectal cancer cell line (ATCC (R); CCL-225) were xenografted subcutaneously as CEACAM5 negative human cells in the left shoulder. This Example was performed at N = 3. After the tumor volume reached approximately 300 mm3, PB009-03 (approximately 20 µg, approximately 120 µCi) was administered intravenously. Photographs were taken by PET 4 hours, 24 hours and 48 hours after administration of PB009-03, and the SUV-Max of the tumor site was measured.

As a result, as shown in Fig. 1A, PB009-03 was shown to accumulate more in LS174T cells (human cells positive for CEACAM5) than in HCT-15 cells (human cells negative for CEACAM5) 4 hours after administration. As shown in Fig. 1B, PB009-03 was found to allow detection of CEACAM5 positive human cancer cells 4 hours to 48 hours after administration.

Example 6: Contrast evaluation of the conjugate in a liver transplant model)

[0131] 1 x 106 LS174T cells expressing luciferase were transplanted into the liver of an immunodeficient mouse (NSG mouse; The Jackson Laboratory) under anesthesia. This example was performed at N = 6. The cell graft in the liver was confirmed as an index of luciferase expression using the IVIS imaging system (PerkinElmer, Inc.). Then, PB009-03 (approximately 14 µg, approximately 100 µCi), prepared in the same manner as in Example 4, was administered intravenously. Photographs were taken with PET 4 hours and 24 hours after administration of PB009-03. The SUV-Max of the liver and the LS174T tumor (which refers to a state in which the transplanted LS174T cells were grafted into the liver) was measured to calculate the ratio between the SUV-Max of the tumor and the SUV-Max of the liver.

As a result, as shown in Fig. 2A, PB009-03 was shown to accumulate in the LS174T tumor grafted to the liver 4 hours after administration.

The signal ratios between the tumor and the liver 4 hours and 24 hours after administration were the values shown in Fig. 2B. From these, it was found that PB009-03 allows the detection of CEACAM5 positive human cancer cells present in the liver 4 hours and 24 hours after administration.

INDUSTRIAL APPLICABILITY

[0132] The Fab fragment of the human anti-CEACAM5 antibody of the present invention and the conjugate comprising the Fab fragment of the human anti-CEACAM5 antibody may be useful in the diagnosis of a cancer selected from the group consisting of colorectal cancer, breast cancer, cancer lung cancer, cancer of the thyroid gland and cancer resulting from their metastasis.

FREE SEQUENCE LISTING TEXT

[0133] The numeric legend <223> in the sequence listing provided below will describe "Artificial Sequence". Specifically, the nucleotide sequences represented by SEQ ID NOs: 1 and 3 of the sequence listing are the nucleotide sequences of the heavy chain and light chain fragments, respectively, from PB009-01. The amino acid sequences represented by SEQ ID NOs: 2 and 4 are the amino acid sequences of the fragment of the heavy chain and the light chain encoded by SEQ ID NOs: 1 and 3, respectively.

Claims (37)

1. Fab fragment of the human anti-CEACAM5 antibody CHARACTERIZED for being selected from the group consisting of the following items (a) and (b): (a) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a region heavy chain variable consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 and a light chain comprising a light chain variable region consisting of an amino acid sequence from amino acid positions 1 to 112 of SEQ ID NO: 4; and (b) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment comprising a heavy chain variable region derived from a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1 to 121 of SEQ ID NO: 2 by modification of glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid, and a light chain comprising a variable region of the light chain consisting of an amino acid sequence from the amino acid positions 1 to 112 of SEQ ID NO: 4. 2. Fab fragment of the human anti-CEACAM5 antibody according to claim 1, CHARACTERIZED by being selected from the group consisting of the following items (a) and (b): (a) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4; and (b) a Fab fragment of the human anti-CEACAM5 antibody comprising a heavy chain fragment derived from a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 by modifying glutamic acid at amino acid position 1 of SEQ ID NO: 2 in pyroglutamic acid and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4. Fab fragment of the human anti-CEACAM5 antibody according to claim 2, CHARACTERIZED by comprising a heavy chain fragment consisting of the amino acid sequence represented by SEQ ID NO: 2 and a light chain consisting of the represented amino acid sequence by SEQ ID NO: 4. 4. Conjugate CHARACTERIZED for comprising a labeling portion and a Fab fragment of the human anti-CEACAM5 antibody defined in any one of claims 1 to 3. 5. Conjugate according to claim 4, CHARACTERIZED by the fact that the labeling portion is (i) a binder and a binding agent, (ii) a binder, (iii) a fluorescent dye and a binding agent, or (iv) a fluorescent dye. 6. Conjugate according to claim 5, CHARACTERIZED by the fact that the labeling portion is (i) a linker and a linker, or (ii) a linker. 7. Conjugate according to claim 6, CHARACTERIZED by the fact that the ligand is a ligand represented by the following formula (A): [Chemical formula 1] (A) wherein the wavy line represents the binding to the Fab fragment of the human anti-CEACAM5 antibody or to the binding agent. 8. Conjugate according to claim 6, CHARACTERIZED by the fact that the labeling portion is (i) a ligand and a binding agent, wherein the ligand and the binding agent are a group represented by the following formula (A '): [Chemical formula 2] (A') where the wavy line represents the binding to the Fab fragment of the human anti-CEACAM5 antibody. 9. Conjugate according to claim 8, CHARACTERIZED by the fact that the Fab fragment of the human anti-CEACAM5 antibody is attached to the carbon atom of a terminal C (= S) group of the labeling portion through an amino group in the Fab fragment. 10. Conjugate according to claim 6, CHARACTERIZED by the fact that it is represented by the following formula (I): (L-X) p-Ab (I) where Ab is the Fab fragment of the human anti-CEACAM5 antibody; L is the ligand; X is the linker or a bond; and p is a natural number from 1 to 25. 11. Conjugate according to claim 10, CHARACTERIZED by the fact that L is a linker represented by the following formula (A): [Chemical formula 3] (A) where the wavy line represents the bond to X (or Ab when X is a bond). 12. Conjugate according to claim 8 or 11, CHARACTERIZED by the fact that it is represented by the following formula (II): [Chemical formula 4] (II) p where Ab is the Fab fragment of the human anti-CEACAM5 antibody; and p is a natural number from 1 to 25, where Ab is attached to the terminal group C (= S) carbon atom of the labeling portion through an amino group on Ab. 13. Conjugate according to any one of claims 10 to 12, CHARACTERIZED by the fact that p is a natural number from 1 to 16. 14. Conjugate according to any one of claims 10 to 12, CHARACTERIZED by the fact that p is a natural number from 4 to 16. Conjugate according to any one of claims 5 to 14, CHARACTERIZED by further comprising a metal. 16. Conjugate, according to claim 15, CHARACTERIZED by the fact that the metal is a metallic radioisotope. 17. Conjugate, according to claim 15, CHARACTERIZED by the fact that the metal is 89Zr. 18. Conjugate according to claim 16 or 17, characterized by being for use as a PET tracer. 19. Polynucleotide CHARACTERIZED because it is selected from the group consisting of the following (a) and (b): (a) a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the defined human anti-CEACAM5 antibody Fab fragment (a) in claim 1; and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1. 20. Polynucleotide CHARACTERIZED for being selected from the group consisting of the following items (a) and (b): (a) a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody defined in claim 3; and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3. 21. Expression vector FEATURED for comprising the following items (a) and / or (b): (a) a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of the defined fragment of the anti-CEACAM5 human antibody (a) in claim 1; and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1. 22. Expression vector CHARACTERIZED for comprising the following items (a) and / or (b): (a) a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody defined in the claim 3; and (b) a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3. 23. Host cell CHARACTERIZED because it is selected from the group consisting of the following items (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence that encodes the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1; (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) defined in claim 1, and a polynucleotide containing a nucleotide sequence which encodes the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1; and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) defined in claim 1, and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1. 24. Host cell CHARACTERIZED for being selected from the group consisting of the following items (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody defined in claim 3; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3; (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment defined in claim 3, and a polynucleotide containing a nucleotide sequence encoding light chain of the Fab fragment of the human anti-CEACAM5 antibody, defined in claim 3; and (d) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment defined in claim 3, and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3. 25. Method for the production of a Fab fragment of the anti-human CEACAM5 antibody, CHARACTERIZED by understanding the step of culturing a host cell selected from the group consisting of the following items (a) to (c) to express the Fab fragment the human anti-CEACAM5 antibody: (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1, and a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) defined in claim 1, and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1; and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment (a) defined in claim 1, and a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody (a) defined in claim 1. 26. Method for the production of a Fab fragment of the anti-human CEACAM5 antibody, CHARACTERIZED by understanding the step of culturing a host cell selected from the group consisting of the following items (a) to (c) to express the Fab fragment of the human anti-CEACAM5 antibody: (a) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of a Fab fragment of the human anti-CEACAM5 antibody defined in claim 3, and one polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3; (b) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment defined in claim 3, and an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3; and (c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the heavy chain fragment of the human anti-CEACAM5 antibody Fab fragment defined in claim 3, and a host cell transformed with a vector of an expression comprising a polynucleotide containing a nucleotide sequence encoding the light chain of the Fab fragment of the human anti-CEACAM5 antibody defined in claim 3. 27. Method for the production of a conjugate comprising a labeling portion and a Fab fragment of the human anti-CEACAM5 antibody, characterized by comprising the steps of: preparing the Fab fragment of the human anti-CEACAM5 antibody by a method defined in claim 25 or 26; and covalently attaching the Fab fragment to the tag portion. 28. Method for the production of a conjugate comprising a linker and a Fab fragment of the human anti-CEACAM5 antibody, characterized by comprising the steps of: preparing the Fab fragment of the human anti-CEACAM5 antibody by a method defined in claim 25 or 26; and covalently attaching the Fab fragment to the linker via a linker or directly. 29. Method for the production of a conjugate comprising a labeling portion labeled with a metal radioisotope and a Fab fragment of the human anti-CEACAM5 antibody, CHARACTERIZED as comprising the steps of: producing a conjugate comprising a ligand and a Fab fragment of the anti-human antibody - human CEACAM5 by a method defined in claim 28; and connecting the metal radioisotope to the conjugate ligand through a coordinated connection. 30. Diagnostic composition CHARACTERIZED for comprising a conjugate defined in any one of claims 15 to 18, and a pharmaceutically acceptable carrier. 31. Composition for diagnosis, according to claim 30, CHARACTERIZED by the fact that it is a drug for clinical staging. 32. Composition for diagnosis, according to claims 30 or 31, CHARACTERIZED because it is for use in the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from their metastasis. 33. Diagnostic composition according to claim 32, CHARACTERIZED for use in the diagnosis of colorectal cancer or cancer resulting from colorectal cancer metastasis. 34. Diagnostic composition according to claim 33, CHARACTERIZED by the fact that the cancer resulting from colorectal cancer metastasis is metastatic liver cancer. 35. Use of a conjugate according to any one of claims 15 to 18, CHARACTERIZED for the production of a composition for the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting metastasis. 36. Conjugate according to any one of claims 15 to 18, CHARACTERIZED for use in the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from their metastasis. 37. Method for the diagnosis of colorectal cancer, breast cancer, lung cancer, cancer of the thyroid gland or a cancer resulting from their metastasis, CHARACTERIZED because it comprises administering a conjugate, defined in any one of claims 15 to 18, to a individual.